Sample preparation and analysis for atmospheric pressure matrix assisted laser desorption ionization time of flight mass spectrometry (AP- MALDI-TOF-MS) was investigated. By investigating the effects that sample preparation has upon MALDI signal, better analysis can be carried out. The influence of sample deposition was studied by not only observing the signal intensity produced but also by quantitation. Isotope dilution mass spectrometry (IDMS) was used for the quantitation of three different analytes. The results indicated that not only was signal greatly affected by sample deposition but the effect on quantitation error was also statistically significant among the three different sample deposition techniques that were evaluated. <br>Components of sample preparation solution were studied using polyethylene glycol (PEG) and polystryrene (PS) of different weights. This study altered the amounts of matrix, analyte and cationizing agent that were used to make up each sample. Not only did the sample signal intensity greatly vary which had statistical significance but a shifting of the polymer sample peaks was also observed. This confirms that sample preparation is of extreme importance for MALDI analysis. <br>Carpet fibers, glutathione and cell wall extracts from the bacteria Staphylococcus Epidermidis were also studied by AP- MADLI-TOF-MS. These analytes were carefully studied to provide an accurate characterization of each. The diversity of the analytes studied highlights the incredible capabilities that MADLI possesses being able to analyze a range of analytes. Though the samples were diverse each one was able to be completely and comprehensively analyzed using AP-MALDI-TOF-MS. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

<p>This essay is written within my education for Technical Officer in the Swedish armed forces. The essay describes the alternative fuels that the Swedish armed forces can make use of if the supply of fossil fuels is insecure. The alternative fuels that are described and compared are; Synthetic diesel, Organic diesel, Methanol and Ethanol. The essay describes the process of producing the alternatives, the need for modification and the economics for each alternative. The alternatives are compared and assessed for their ability to secure the supply of fuel to the Swedish Armed Forces. The result of the study is that, while synthetic diesel has the best chemical constitution but is not produced in Sweden, the best alternative fuel for the Swedish armed forces at this moment is organic diesel.</p>

The emerging field of synthetic biology aims to use artificially designed genetic circuits to program living cells, much as engineers program a computer or control electronic or mechanical systems. This thesis focuses on the design and implementation of synthetic gene circuits in the bacterium Escherichia coli to create new cellular functions, and on the quantitative characterization and modelling of these circuits. Though important in any engineering discipline, quantitative system characterization has been poorly explored in synthetic biology. We have performed a quantitative system characterization by implementing simple gene circuits in Escherichia coli. The work showed that the level and variability of gene expression varied across different cell strains, and we investigated how these effects manifested through the coupled effects of cell division, cellular growth rate, and plasmid copy number regulation. The work suggests that gene circuit modules from a standard library cannot be used universally; the cellular context and the time dependent dynamics must be considered when implementing gene circuits. In order to work precisely as engineering devices, synthetic gene circuits must be appropriately tuned. One standard method of tuning genetic circuits requires altering the DNA sequences by extensive molecular biology work. Part of this thesis focuses on developing easily tunable gene circuits. A set of circuits were developed in E. coli where the shape of the chemically induced signal response curves can be tuned from a band structure to a sigmoidal structure simply by altering the temperature in a single system. Another set of circuits was developed which demonstrate a range of chemically tunable signal response curves along with multiple functions in a single device. One of the ultimate goals of synthetic biology is to program living cell in a human-controlled way. To this end, I developed a set of genetic devices that could work as ‘in cell disease prevention devices,’ preventing an otherwise fatal viral infection in E. coli. The device displays a number of ‘device’ properties: being dormant under normal conditions, detecting the onset of the disease state, turning on automatically to prevent a lethal outcome, and being subject to external deactivation when desired. The combination of design, characterization, and mathematical understanding explored in this work represents a contribution in the direction of developing synthetic biology as a well-founded engineering discipline.

<p>Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism <i>Saccharomyces cerevisiae</i>, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2Ä, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and á2 proteins encoded by the mating type genes MATa and MATá, respectively. Diploid cells heterozygous for mating type (a/á) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability.</p>

This is the first systematic experimental investigation of partitioning of Br between apatites and coexisting melts and the uptake of Br by scapolite and sodalite. Twenty-nine partitioning experiments between fluorapatite (FAP) /chlorapatite (ClAP) and coexisting melts were conducted in the system of CaO-P2O5-CaF2-CaCl2NaBr at 1120 degree C to 1400 degree C and atmospheric pressure. The partition coefficients (D) with errors of 1 sigma in parentheses are as follow: <p>DF (ClAP/melt) 3.59(64) at 1120 degree C to 4.13(22) at 1330 degree C <p> DF (FAP/melt) 1.05(4) at 1220 degree C to 1.07 at 1400 degree C <p> DCl (ClAP/melt) 1.07(1) at 1120 degree C to 0.83 at 1330 degree C <p> DCl (FAP/melt) 0.127(2) at 1250 degree C to 0.115 at 1400 degree C <p> DBr (ClAP/melt) 0.32(9) at 1120 degree C to 0.42(5) at 1330 degree C <p> DBr (FAP/melt) 0.020(3) at 1220 degree C to 0.016 at 1400 degree C <p> Seven exchange experiments at one atmospheric pressure and 800 to 1000 degree C yield the following distribution coefficients for Br-Cl exchanges between marialitic scapolite or sodalite and coexisting hydrous NaCl-NaBr melts: KD (marialite-melt) = 0.92 +/- 0.10 and KD (sodalite-melt) = 1.18 +/- 0.10. Therefore, the Cl/Br values in marialitic scapolite and sodalite closely reflect the halogen proportions of their coexisting melts or fluids. <p> The second part of this thesis project analyzes the halogen (F, Cl, Br) contents in natural fluorapatite and scapolite by X-ray fluorescence microprobe (XRF) for Br and electron microprobe (EMPA) for other elements. All selected localities and environments are interesting, because the origins of the parental fluids/melts are controversial. The halogen compositions of 29 natural apatite grains from the Aoshan fluorapatite-magnetite deposit (China), the Oka carbonatite complex (Quebec), and Chinese mantle xenoliths and 36 scapolite samples from the Tieshan Fe-Cu skarn deposit (China), the Nickel Plate gold deposit (British Columbia), and the Grenville pegmatite/skarn/vein deposits (Ontario and Quebec) have been analyzed by electron microprobe (EMPA) and X-ray fluorescence microprobe (XRF). Twenty six whole-rock samples from the Aoshan deposit have also been analyzed by XRF for major and trace elements. <p> Fluorapatite from the Aoshan fluorapatite-magnetite deposit is Cl-bearing with 0.38-0.98 wt% Cl, 1.83-3.45 wt% F, and 0-52 ppm Br. Fluorapatite from Chinese mantle xenoliths has similar halogen compositions to the Aoshan fluorapatite. Fluorapatite from the Oka carbonatite has trace amounts of chlorine (up to 0.052 wt%) and bromine (from 9 to 57 ppm). Applications for the experimental results suggest that the Aoshan Fe-Cu deposit has Cl/Br values comparable to those of mantle sources and that the anomalously low Cl/Br values in Oka fluorapatite require Br-enriched sources. <p> The Cl/Br values (weight) of marialitic scapolite from the Tieshan Fe-Cu deposit cluster around 626 +/- 92, supporting an origin involving hydrothermal brines from associated evaporites. Scapolite-group minerals in the exoskarns of the Nickel Plate Au skarn deposit have Cl/Br from 560 to 570, higher than those (110 to 180) of their counterparts in the endoskarns and vuggy cavities. This variation is attributable to an increased involvement of magmatic water from distal to proximal zones. Similarly, scapolite-group minerals in the Grenvillian U-Th-Mo-REE pegmatite-skarn-vein deposits vary widely in Cl/Br from 80 to 380, indicative of mixed sources of hydrothermal fluids from magmatic sources and from associated sedimentary rocks. <p> The experimentally determined partition coefficients of halogens between minerals (apatites, scapolite, and sodalite) and fluids/melts of this study have wide applications in the interpretation of source and evolution of hydrothermal fluids in mineralization processes and other geological systems. Applications of those partition coefficients to selected mineral deposits and mantle xenoliths confirm their significance.

The emerging field of synthetic biology aims to use artificially designed genetic circuits to program living cells, much as engineers program a computer or control electronic or mechanical systems. This thesis focuses on the design and implementation of synthetic gene circuits in the bacterium Escherichia coli to create new cellular functions, and on the quantitative characterization and modelling of these circuits. Though important in any engineering discipline, quantitative system characterization has been poorly explored in synthetic biology. We have performed a quantitative system characterization by implementing simple gene circuits in Escherichia coli. The work showed that the level and variability of gene expression varied across different cell strains, and we investigated how these effects manifested through the coupled effects of cell division, cellular growth rate, and plasmid copy number regulation. The work suggests that gene circuit modules from a standard library cannot be used universally; the cellular context and the time dependent dynamics must be considered when implementing gene circuits. In order to work precisely as engineering devices, synthetic gene circuits must be appropriately tuned. One standard method of tuning genetic circuits requires altering the DNA sequences by extensive molecular biology work. Part of this thesis focuses on developing easily tunable gene circuits. A set of circuits were developed in E. coli where the shape of the chemically induced signal response curves can be tuned from a band structure to a sigmoidal structure simply by altering the temperature in a single system. Another set of circuits was developed which demonstrate a range of chemically tunable signal response curves along with multiple functions in a single device. One of the ultimate goals of synthetic biology is to program living cell in a human-controlled way. To this end, I developed a set of genetic devices that could work as ‘in cell disease prevention devices,’ preventing an otherwise fatal viral infection in E. coli. The device displays a number of ‘device’ properties: being dormant under normal conditions, detecting the onset of the disease state, turning on automatically to prevent a lethal outcome, and being subject to external deactivation when desired. The combination of design, characterization, and mathematical understanding explored in this work represents a contribution in the direction of developing synthetic biology as a well-founded engineering discipline.

Architectural research for Field-Programmable Gate Arrays (FPGAs) tends to use an experimental approach. The benchmark circuits are used not only to compare different architectures, but also to ensure that the FPGA is sufficiently flexible to implement the desired variety of circuits. The most common benchmark circuits used for architectural research are circuits from the Microelectronics Center of North Carolina (MCNC). These circuits are small; they occupy less than 3% [5] of the largest available commercial FPGA. Moreover, these circuits are more representative of the glue logic circuits that were targets of early devices. This contrasts with the trend towards implementing Systems on Chip (SoCs) on FPGAs where several functional modules are integrated into a single circuit which is mapped onto one device. In this thesis, we develop a synthetic system-level circuit generator that connects pre-existing circuits in a realistic manner to build large netlists that share the characteristics of real SoC circuits. This generator is based on a survey of contemporary circuit designs from industrial and academic sources. We demonstrate that these system-level circuits scale well and that their post-routing characteristics match the results of large pre-existing benchmarks better than the results of circuits from previous synthetic generators.

This essay is written within my education for Technical Officer in the Swedish armed forces. The essay describes the alternative fuels that the Swedish armed forces can make use of if the supply of fossil fuels is insecure. The alternative fuels that are described and compared are; Synthetic diesel, Organic diesel, Methanol and Ethanol. The essay describes the process of producing the alternatives, the need for modification and the economics for each alternative. The alternatives are compared and assessed for their ability to secure the supply of fuel to the Swedish Armed Forces. The result of the study is that, while synthetic diesel has the best chemical constitution but is not produced in Sweden, the best alternative fuel for the Swedish armed forces at this moment is organic diesel.

This is the first systematic experimental investigation of partitioning of Br between apatites and coexisting melts and the uptake of Br by scapolite and sodalite. Twenty-nine partitioning experiments between fluorapatite (FAP) /chlorapatite (ClAP) and coexisting melts were conducted in the system of CaO-P2O5-CaF2-CaCl2NaBr at 1120 degree C to 1400 degree C and atmospheric pressure. The partition coefficients (D) with errors of 1 sigma in parentheses are as follow: <p>DF (ClAP/melt) 3.59(64) at 1120 degree C to 4.13(22) at 1330 degree C <p> DF (FAP/melt) 1.05(4) at 1220 degree C to 1.07 at 1400 degree C <p> DCl (ClAP/melt) 1.07(1) at 1120 degree C to 0.83 at 1330 degree C <p> DCl (FAP/melt) 0.127(2) at 1250 degree C to 0.115 at 1400 degree C <p> DBr (ClAP/melt) 0.32(9) at 1120 degree C to 0.42(5) at 1330 degree C <p> DBr (FAP/melt) 0.020(3) at 1220 degree C to 0.016 at 1400 degree C <p> Seven exchange experiments at one atmospheric pressure and 800 to 1000 degree C yield the following distribution coefficients for Br-Cl exchanges between marialitic scapolite or sodalite and coexisting hydrous NaCl-NaBr melts: KD (marialite-melt) = 0.92 +/- 0.10 and KD (sodalite-melt) = 1.18 +/- 0.10. Therefore, the Cl/Br values in marialitic scapolite and sodalite closely reflect the halogen proportions of their coexisting melts or fluids. <p> The second part of this thesis project analyzes the halogen (F, Cl, Br) contents in natural fluorapatite and scapolite by X-ray fluorescence microprobe (XRF) for Br and electron microprobe (EMPA) for other elements. All selected localities and environments are interesting, because the origins of the parental fluids/melts are controversial. The halogen compositions of 29 natural apatite grains from the Aoshan fluorapatite-magnetite deposit (China), the Oka carbonatite complex (Quebec), and Chinese mantle xenoliths and 36 scapolite samples from the Tieshan Fe-Cu skarn deposit (China), the Nickel Plate gold deposit (British Columbia), and the Grenville pegmatite/skarn/vein deposits (Ontario and Quebec) have been analyzed by electron microprobe (EMPA) and X-ray fluorescence microprobe (XRF). Twenty six whole-rock samples from the Aoshan deposit have also been analyzed by XRF for major and trace elements. <p> Fluorapatite from the Aoshan fluorapatite-magnetite deposit is Cl-bearing with 0.38-0.98 wt% Cl, 1.83-3.45 wt% F, and 0-52 ppm Br. Fluorapatite from Chinese mantle xenoliths has similar halogen compositions to the Aoshan fluorapatite. Fluorapatite from the Oka carbonatite has trace amounts of chlorine (up to 0.052 wt%) and bromine (from 9 to 57 ppm). Applications for the experimental results suggest that the Aoshan Fe-Cu deposit has Cl/Br values comparable to those of mantle sources and that the anomalously low Cl/Br values in Oka fluorapatite require Br-enriched sources. <p> The Cl/Br values (weight) of marialitic scapolite from the Tieshan Fe-Cu deposit cluster around 626 +/- 92, supporting an origin involving hydrothermal brines from associated evaporites. Scapolite-group minerals in the exoskarns of the Nickel Plate Au skarn deposit have Cl/Br from 560 to 570, higher than those (110 to 180) of their counterparts in the endoskarns and vuggy cavities. This variation is attributable to an increased involvement of magmatic water from distal to proximal zones. Similarly, scapolite-group minerals in the Grenvillian U-Th-Mo-REE pegmatite-skarn-vein deposits vary widely in Cl/Br from 80 to 380, indicative of mixed sources of hydrothermal fluids from magmatic sources and from associated sedimentary rocks. <p> The experimentally determined partition coefficients of halogens between minerals (apatites, scapolite, and sodalite) and fluids/melts of this study have wide applications in the interpretation of source and evolution of hydrothermal fluids in mineralization processes and other geological systems. Applications of those partition coefficients to selected mineral deposits and mantle xenoliths confirm their significance.

<p>Both prokaryotic and eukaryotic cells are equipped with DNA repair mechanisms to protect the integrity of their genome in case of DNA damage. In the eukaryotic organism <i>Saccharomyces cerevisiae</i>, MMS2 encodes a ubiquitin-conjugating enzyme variant protein belonging to the RAD6 repair pathway; MMS2 functions in error-free postreplication repair (PRR), a subpathway parallel to REV3 mutagenesis. A mutation in MMS2 does not result in extreme sensitivity to DNA damaging agents; however, deletion of both subpathways of PRR results in a synergistic phenotype. By taking advantage of the synergism between error-free PRR and mutagenesis pathway mutations, a conditional synthetic lethal screen was used to identify novel genes genetically involved in PRR. A synthetic lethal screen was modified to use extremely low doses of MMS that would not affect the growth of single mutants, but would effectively kill the double mutants. Fifteen potential mutants were characterized, of which twelve were identified as known error-prone PRR genes. Characterization of mutations in strains SLM-9 and SLM-11, that are conditionally synthetically lethal with mms2Ä, revealed functions for both checkpoints and mating-type heterozygosity in regulating PRR. Cell cycle checkpoints monitor the integrity of the genome and ensure that cell cycle progression is deferred until chromosome damage is repaired. The checkpoint genes genetically interact with both the error-free and error-prone branches of PRR, potentially for delaying cell cycle progression to allow time for DNA repair, and for signaling the stage of the cell cycle and thus DNA content. Other potential monitors for DNA content are the a1 and á2 proteins encoded by the mating type genes MATa and MATá, respectively. Diploid cells heterozygous for mating type (a/á) show an increased resistance to UV damage and are more recombination-proficient than haploid cells. Haploid PRR mutants expressing both mating type genes show an increased resistance to DNA-damaging agents. This phenomenon is specific to PRR: it was not seen in excision repair-deficient and recombination-deficient mutants tested. The rescuing effect seen in PRR mutants heterozygous for mating type is likely the result of channeling lesions into a recombination repair pathway and away from the non-operational PRR pathway. Both checkpoint and mating type genes play a role in regulating PRR. Almost certainly these genes are required to monitor the cell cycle stage and DNA content to determine the best mechanism to repair the damaged DNA thus preventing genomic instability.</p>